A Developmental Stage-Specific Switch from DAZL to
BOLL Occurs during Fetal Oogenesis in Humans, but Not
Mice
Jing He1, Kayleigh Stewart1, Hazel L. Kinnell1, Richard A. Anderson1, Andrew J. Childs1,2*
1 MRC Centre for Reproductive Health, the Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, United Kingdom, 2 Department of
Comparative Biomedical Sciences, the Royal Veterinary College, University of London, Camden, London, United Kingdom
Abstract
The Deleted in Azoospermia gene family encodes three germ cell-specific RNA-binding proteins (DAZ, DAZL and
BOLL) that are essential for gametogenesis in diverse species. Targeted disruption of Boll in mice causes malespecific spermiogenic defects, but females are apparently fertile. Overexpression of human BOLL promotes the
derivation of germ cell-like cells from genetically female (XX), but not male (XY) human ES cells however, suggesting
a functional role for BOLL in regulating female gametogenesis in humans. Whether BOLL is expressed during
oogenesis in mammals also remains unclear. We have therefore investigated the expression of BOLL during fetal
oogenesis in humans and mice. We demonstrate that BOLL protein is expressed in the germ cells of the human fetal
ovary, at a later developmental stage than, and almost mutually-exclusive to, the expression of DAZL. Strikingly,
BOLL is downregulated, and DAZL re-expressed, as primordial follicles form, revealing BOLL expression to be
restricted to a narrow window during fetal oogenesis. By quantifying the extent of co-expression of DAZL and BOLL
with markers of meiosis, we show that this window likely corresponds to the later stages of meiotic prophase I.
Finally, we demonstrate that Boll is also transiently expressed during oogenesis in the fetal mouse ovary, but is
simultaneously co-expressed within the same germ cells as Dazl. These data reveal significant similarities and
differences between the expression of BOLL homologues during oogenesis in humans and mice, and raise questions
as to the validity of the Boll-/- mouse as a model for understanding BOLL function during human oogenesis.
Citation: He J, Stewart K, Kinnell HL, Anderson RA, Childs AJ (2013) A Developmental Stage-Specific Switch from DAZL to BOLL Occurs during Fetal
Oogenesis in Humans, but Not Mice. PLoS ONE 8(9): e73996. doi:10.1371/journal.pone.0073996
Editor: Stefan Schlatt, University Hospital of Münster, Germany
Received June 6, 2013; Accepted July 29, 2013; Published September 25, 2013
Copyright: © 2013 He et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Medical Research Foundation [grant 354FRG to AJC] and the Medical Research Council [grant G1100357 to RAA].
J.H. is supported by a University of Edinburgh Darwin Scholarship and K.S. is the recipient of a Society for Reproduction and Fertility Summer Vacation
Scholarship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: [email protected]
Introduction
The prototypic DAZ gene is present in multiple copies on the
Y chromosome of humans and Old World monkeys [12], and
was identified as a candidate male factor infertility gene from its
location in the AZFc region of the Y chromosome, an area
frequently deleted in men with severe oligozoospermia or
azoospermia [13]. DAZ arose from a duplication of the
autosomal homologue Dazl (Deleted in azoospermia-like) [14],
the expression of which is required at multiple stages of germ
cell development in male and female mammals [1,11,15-20].
BOLL (also known as BOULE, or BOULE-LIKE), is the
ancestral member of the family, with orthologues throughout
the metazoa [21,22]. Boule-deficient flies display defective
spermatogenesis [3], arising from a failure to translate mRNA
encoding the CDC25 homologue Twine, resulting in meiotic
arrest [8]. Targeted disruption of Boll in mice results in malespecific infertility [22], due to arrest of spermiogenesis at the
round spermatid stage [4]. Boll-deficient female mice are
The Deleted in Azoospermia (DAZ) gene family encodes
three conserved RNA-binding proteins (DAZ, DAZL and BOLL),
the expression of which is largely restricted to germ cells. DAZfamily proteins are essential for germ cell development in
diverse organisms, and inactivating mutations in members of
the DAZ family result in a failure of germ cell development in
either or both sexes [1-4]. DAZ-family proteins are
characterised by a conserved RRM-type RNA binding domain,
and a unique 24 amino-acid DAZ repeat sequence, and act as
regulators of mRNA translation [5], mRNA transport [6] and
mRNA metabolism during cellular stress [7]. The sterility
phenotypes observed in DAZ-family protein-deficient
organisms are believed to arise from a failure to translate key
mRNAs required for germ cell development [5,8-11].
PLOS ONE | www.plosone.org
1
September 2013 | Volume 8 | Issue 9 | e73996
DAZL and BOLL during Human and Mouse Oogenesis
Distinct spatio-temporal distributions of DAZL and
BOLL proteins during human fetal oogenesis
apparently fertile however, leading to the hypothesis that Boll is
dispensable for mammalian oogenesis [4,22]. Intriguingly
however, overexpression of human BOLL can enhance the
derivation of primordial germ cell-like cells from genetically
female (XX) but not male (XY) human embryonic stem cells
[23], suggesting a potential functional role for BOLL in human
female germ cell development that remains to be established.
Although Boll transcripts have been reported in the
mammalian fetal ovary [22,24,25] the existence of Boll protein
in mammalian female germ cells has not yet been
demonstrated, and it has been suggested that Boll protein may
never be produced in the mammalian female germline [25]. We
have previously observed a period of human female germ cell
development, prior to primordial follicle formation, during which
DAZL expression was reduced [26], and have therefore
undertaken the first detailed study of BOLL expression during
oogenesis in the fetal mammalian (human and mouse) ovary,
and compared this with the expression of DAZL. We
demonstrate for the first time that BOLL protein is expressed in
the human female fetal germline, and that the expression of
DAZL and BOLL is largely non-overlapping, with each protein
expressed in a distinct population of germ cells at different
developmental stages. Additionally, we show that Boll is also
transiently expressed in the germ cells of the fetal mouse
ovary, but in striking contrast to the human, shows extensive
co-expression in the same germ cells as Dazl. Together, these
data indicate that BOLL may have specific and important
functions in regulating oogenesis in humans - distinct from
those of DAZL - in contrast to its dispensability in mice.
The expression and distribution of DAZL protein in the
human fetal ovary has been reported previously [26]. However
the expression of BOLL protein in the mammalian ovary has
not previously been reported, and it has been proposed that it
is not expressed [25], despite the presence of transcript
[22,24]. We therefore sought to establish whether BOLL protein
is expressed in the human fetal ovary, and if so, what
relationship it has to the expression of DAZL.
We confirmed the specificity of the DAZL and BOLL
antibodies used in this study (listed in Table S3) by performing
immunofluorescence on HEK293 cells transfected with vectors
expression either Myc- and FLAG-tagged human DAZL or
BOLL (Figure S1). Anti-DAZL antibodies bound epitopes only
in cells transfected with DAZL expression constructs, and not in
untransfected cells or cells transfected with a BOLL expression
vector. Conversely, the anti-BOLL antibody detected signals in
BOLL-expressing cells, but not in cells ectopically-expressing
DAZL or untransfected controls. We further validated the
specificity of the antibodies by performing western blotting on
lysates of DAZL and BOLL-transfected HEK293 cells. The antiDAZL antibodies detected bands of appropriate sizes only in
lysates of DAZL-expressing HEK293 cells, and not in those of
BOLL-transfected or untransfected cells (Figure S2A,B).
Similarly, the anti-BOLL antibody detected a band only in
extracts of BOLL-transfected cells (Figure S2C).
Using single immunofluorescence we were able to detect
DAZL protein in the germ cells of 8-9wga human fetal ovaries,
with the protein localising to both germ cell nuclei and
cytoplasm as reported previously [26,27]. We were unable to
detect BOLL protein, consistent with our inability to detect
BOLL mRNA at this stage (Figure 2A). Both DAZL and BOLL
were expressed in germ cells in the second trimester, and dual
immunofluorescence analysis revealed that their expression
patterns are distinct and dynamic (Figure 2B). At 14wga, most
germ cells were DAZL-positive, with relatively few expressing
BOLL. By 18wga, BOLL-positive cells were more abundant,
although DAZL-positive germ cells were still readily detectable.
Notably, we found that the oocytes of primordial follicles at
20wga expressed DAZL, but not BOLL, suggesting oocytes
downregulate BOLL and re-express DAZL at or around the
time of follicle formation. In all specimens examined, only few
germ cells showed expression of both DAZL and BOLL (Figure
2B and C).
We noted distinct spatial distributions of DAZL and BOLL in
the human fetal ovary. Consistent with earlier studies [26], we
found DAZL-expressing germ cells to be predominantly
localised towards the periphery of the ovary, the site of less
mature germ cells (Figure 2C). In contrast, BOLL-positive germ
cells were localised more towards the central medullary region,
which contains the more mature germ cells, and where
primordial follicles are first formed. Germ cells expressing both
DAZL and BOLL were rare (Figure 2B and C), suggesting
DAZL and BOLL mark distinct populations of germ cells.
Together, these data reveal that BOLL is indeed expressed in
germ cells of the human fetal ovary, and at a later stage of
germ cell development than DAZL. Furthermore, the
Results
DAZL and BOLL show overlapping, but distinct,
patterns of gene expression during human fetal ovary
development
We first investigated the expression of DAZL and BOLL
during human fetal ovary development, using qRT-PCR (Figure
1A, B). Previous work revealed DAZL to be upregulated
between the first and second trimesters of human fetal ovarian
development [26], however the second trimester encompasses
two key developmental stages; the formation of germ cell nests
and entry into and progression through meiotic prophase I
(approximately 12-16 weeks gestational age (wga)) and the
breakdown of germ cell nests and the assembly of individual
oocytes into primordial follicles (from around 17wga onwards).
We therefore sought to establish at which of these
developmental stages the increase in DAZL occurs, by dividing
the second trimester into early (14-16wga) and late
(17-20wga), and compare this to the expression of BOLL. In
the first trimester (8-9wga), DAZL expression is low and BOLL
undetectable. In the early second trimester (14-16wga) both
DAZL expression significantly increases, and BOLL mRNA first
becomes detectable (p<0.001). At both 14-16 and 17-20wga
weeks BOLL mRNA levels were lower than that of DAZL,
relative to the internal housekeeping control RPL32. Consistent
with previous immunohistochemical studies [21] we were
unable to detect BOLL mRNA in the human fetal testis across
this developmental window (data not shown).
PLOS ONE | www.plosone.org
2
September 2013 | Volume 8 | Issue 9 | e73996
DAZL and BOLL during Human and Mouse Oogenesis
Figure 1. Distinct patterns of DAZL and BOLL expression during human ovary development. A) DAZL transcript levels
increase significantly from 8-9 to 14-16 weeks, with no significant change thereafter. B) BOLL expression is first detected at 14-16
weeks gestation, and but does not change significantly by 17-20 weeks (n=5-6, ***p<0.001 vs 8-9 weeks for both DAZL and BOLL).
doi: 10.1371/journal.pone.0073996.g001
PLOS ONE | www.plosone.org
3
September 2013 | Volume 8 | Issue 9 | e73996
DAZL and BOLL during Human and Mouse Oogenesis
Figure 2. Dynamic changes of DAZL and BOLL protein expression during development of the human fetal ovary. (A) at
65d gestation, DAZL is expressed in the nuclei and cytoplasm of germ cells in the human fetal ovary, but BOLL is not detected (B)
At 14, 18 and 20 weeks gestation, DAZL is detected only in germ cell cytoplasm (arrows). BOLL-positive germ cells (arrowheads)
are detectable from 14 weeks onwards, and increase in abundance with increasing gestation. Rare double-positive cells are marked
by the asterisk. Primordial follicles at 20 weeks gestation (arrows, bottom left panel) express only DAZL. C) Tiled image of 18 weeks
gestation fetal ovary section showing minimal co-localisation of DAZL and BOLL. DAZL is expressed in less mature germ cells in a
more peripheral localisation, while BOLL predominantly expressed in more mature, centrally-located germ cells. Scale bar (A) and
(B) 20 µm, (C) 50µm.
doi: 10.1371/journal.pone.0073996.g002
PLOS ONE | www.plosone.org
4
September 2013 | Volume 8 | Issue 9 | e73996
DAZL and BOLL during Human and Mouse Oogenesis
expression of BOLL in germ cells prior to, but not following,
follicle formation suggests that BOLL is expressed in a narrow
developmental window, subsequent to DAZL expression in
early, less mature germ cells, and before DAZL expression is
reactivated in primordial follicles.
Telangiectasia Mutated (phospho-ATM) protein, which is
expressed by germ cells from pre-leptotene to pachytene of
meiotic prophase I [30]. Consistent with the pattern seen with
SYCP3, we found that a significantly greater proportion of
phospho-ATM-positive germ cells also expressed BOLL (78%)
than expressed DAZL (37%; p<0.05) (Figure S3A,B). Together,
these data suggest that DAZL is expressed before, and
persists into, early meiotic prophase I, but is down-regulated
around the leptotene/zygotene stage and replaced by BOLL.
When the cell reaches the diplotene stage and meiotic arrest
and follicle formation are initiated, BOLL expression is
extinguished and DAZL expression reactivated.
BOLL is expressed by germ cells at a later stage of
development than DAZL
The above data led us to hypothesise that BOLL is
expressed in human fetal ovarian germ cells that have reached
a later stage of development than those expressing DAZL. To
test this, we compared the nuclear diameters of germ cells in
the second trimester human ovary that expressed either DAZL
or BOLL. Germ cell nuclear diameter provides an index of germ
cell maturation, increasing as germ cells progress through
development in the human fetal ovary [28]. We found the
average nuclear diameter of BOLL-positive germ cells to be
significantly greater than that of DAZL-positive germ cells at
14wga (6.60+/-0.12µm vs 9.37+/-0.20µm; p<0.001) and 18wga
(6.87+/-0.12µm vs 9.23+/-0.19µm; p<0.001, n=108-191 per
group; Figure 3A). Furthermore, DAZL- and BOLL-positive
germ cells displayed distinct, although overlapping, nuclear
diameter frequency distributions (Figure 3B and C); with most
DAZL-positive germ cells distributed between at 4 to 8µm,
compared to 8 to 12µm for BOLL-positive germ cells (DAZLpositive oocytes in primordial follicles were excluded from this
analysis). Together, these data confirm that BOLL is expressed
by germ cells at a later stage of development than those
expressing DAZL.
The switching of expression from DAZL to BOLL during
fetal oogenesis is not conserved between humans and
mice
Given the striking switch between DAZL and BOLL
expression during human fetal ovarian germ cell development,
the lack of extensive co-expression of both proteins within the
same germ cells, and the apparent lack of an oogenesis defect
in Boll-deficient mice, we sought to establish whether the
expression patterns of DAZL and BOLL are conserved during
fetal oogenesis in humans and mice. We therefore performed
single-antigen immunofluorescence to detect either Dazl or Boll
on sections of fetal mouse ovaries at embryonic day (e) 13.5,
e15.5, e18.5 and postnatal day 0 (P0; Figure 5A), which
correspond approximately to meiosis initiation (e13.5),
leptotene/zygotene (e15.5), pachytene (e18.5) and diplotene/
follicle formation (P0) [31].
Consistent with previous studies, germ cells in the fetal
mouse ovary expressed Dazl at all stages examined (Figure
5A). At e13.5, small groups of Dazl-positive germ cells were
readily detectable in the fetal mouse ovary, and became more
homogeneously distributed throughout the ovary by e15.5. At
e18.5 and P0, Dazl-positive germ cells were clearly larger than
at previous stages, and predominantly localised towards the
periphery of the ovary (Figure 5A). In contrast, Boll was
undetectable at e13.5, 18.5 and P0, but readily detectable in
large numbers of germ cells at e15.5, with a distribution similar
to that of Dazl (Figure 5A).
The distribution of Dazl and Boll in the germ cells of the fetal
mouse ovary at e15.5 (Figure 5A) suggested that Dazl and Boll
may be extensively co-expressed in the same germ cells
during fetal mouse oogenesis. We therefore performed dualimmunofluorescence to detect Dazl and Boll on sections of
e15.5 fetal mouse ovaries (Figure 5B). In stark contrast to our
findings in the human fetal ovary, we detected extensive colocalisation of Dazl and Boll proteins in the germ cells of the
e15.5 fetal mouse ovary. As positive control, we performed
dual-immunofluorescence for both proteins on sections of the
adult mouse testis (a tissue in which both proteins are known to
be expressed), revealing distinct but overlapping patterns of
Dazl and Boll expression, with the latter expressed in germ
cells at a later stage of development than those expressing
Dazl (Figure 5C, far right panel [4,21]). These data reveal the
existence of a conserved, transient, window of BOLL
expression during fetal oogenesis in both humans and mice,
which probably corresponds to a specific stage of meiotic
DAZL and BOLL differentially associate with markers of
meiosis
Between 11wga and 20wga, germ cells in the fetal ovary
form syncytial nests and enter the first meiotic prophase,
arresting at the diplotene stage (at which they remain until
immediately before ovulation). To establish if the timing of the
switch from DAZL to BOLL during human fetal oogenesis
occurs at a specific stage of the first meiotic prophase, we
performed triple immunofluorescence analysis for DAZL, BOLL
and the meiosis marker Synaptonemal Complex Protein 3
(SYCP3; a component of synaptonemal complex which is
expressed from pre-meiosis to diplotene in human fetal ovarian
germ cells [29]). We detected relatively few SYCP3+ germ cells
that also expressed DAZL (Figure 4A,B). In contrast, almost all
BOLL-positive germ cells also expressed SYCP3 at 14, 15 and
17wga (Figure 4A,B), consistent with BOLL being expressed in
germ cells that have reached a later stage of meiosis I than
those expressing DAZL.
Quantification of this confirmed the extensive overlap of
BOLL and SYCP3 expression (Figure 4C); 69% SYCP3positive germ cells also expressed BOLL, a fraction
significantly higher than the 14% of SYCP3-positive germ cells
that also expressed DAZL, 11% of SYCP3-positive germ cells
which expressed both DAZL and BOLL, and the 6% of SYCP3positive germ cells that express neither DAZL or BOLL
(SYCP3-positive only; p<0.001). We also compared the extent
of co-expression of DAZL and BOLL with another meiosis
marker, the phosphorylated isoform of the Ataxia
PLOS ONE | www.plosone.org
5
September 2013 | Volume 8 | Issue 9 | e73996
DAZL and BOLL during Human and Mouse Oogenesis
Figure 3. BOLL-positive germ cells are at a later developmental stage than those expressing DAZL. A) At 14 and 18 weeks
gestation, the average nuclear diameter (an index of germ cell maturation) of BOLL+ cells is larger than that of DAZL+ cells. B) and
C) The nuclear diameter distribution of DAZL and BOLL+ cells at 14 and 18 weeks gestation respectively showing distinct size
distributions for the two proteins. Primordial follicles were excluded from this analysis (n=108-191 germ cells, ***p<0.001)..
doi: 10.1371/journal.pone.0073996.g003
PLOS ONE | www.plosone.org
6
September 2013 | Volume 8 | Issue 9 | e73996
DAZL and BOLL during Human and Mouse Oogenesis
Figure 4. BOLL displays greater co-localisation with the meiosis marker SYCP3 than DAZL. A) Triple immunofluorescence
analysis of DAZL (green), BOLL (blue) and SYCP3 (red) in 14 and 17 week ovary. All BOLL+ germ cells also express SYCP3
(arrowheads), but only a few DAZL+ germ cell express SYCP3 (arrows). Unfilled arrows indicate the SYCP3-positive cells that
express neither DAZL nor BOLL. The asterisk indicates a germ cell nest containing BOLL + SYCP3+, DAZL + SYCP3+ and DAZL
+SYCP- germ cells in close proximity. Scale bars: 20µm. B) magnified image of germ cell nest marked with asterisk in A, showing
neighboring germ cells expressing different combinations of DAZL, BOLL and SYCP3 expression; arrow denotes DAZL + SYCP3+
germ cell, arrowhead denotes BOLL + SYCP+ germ cell. C) Quantification of DAZL, BOLL and SYCP3 co-expression. ~69% of
SYCP3+ cells also express BOLL+. This is significantly greater than the percentage of all the other co-expression patterns (n=3
14-17 week human fetal ovaries; ***p<0.001)..
doi: 10.1371/journal.pone.0073996.g004
PLOS ONE | www.plosone.org
7
September 2013 | Volume 8 | Issue 9 | e73996
DAZL and BOLL during Human and Mouse Oogenesis
Figure 5. Dazl and Boll proteins display distinct patterns of expression during mouse fetal ovary development. A) Single
immunostaining for Dazl or Boll in e13.5, e15.5, e18.5 and P0 mouse ovary. Dazl is highly expressed in germ cells through all
stages of development, whilst Boll is widely expressed in germ cells at e15.5 but not detectable at earlier or later gestations. B)
Double immunostaining for Dazl and Boll in e15.5 mouse fetal ovary: Dazl and Boll show extensive co-expression in germ cells at
this gestation. C) Control showing partial co-localisation of Dazl and Boll in germ cells in the adult mouse testis, an organ known to
express both proteins (arrows). Scale bars: 20µm.
doi: 10.1371/journal.pone.0073996.g005
by distinct populations of germ cells at different stages of
maturation. We find a progressive pattern of expression as
germ cells mature, with DAZL expressed in germ cells prior to,
and in the early stages of meiotic prophase I, after which it is
down-regulated and BOLL expressed. BOLL in turn is also
down-regulated and DAZL re-expressed at the time that germ
cells in the human fetal ovary form primordial follicles (Figure
6A). This represents the first demonstration of BOLL protein
expression during oogenesis in any mammalian species, and
reveals that BOLL is expressed only during a narrow
developmental window, which appears to correspond to mid-to-
prophase I. However, the striking switch from DAZL expression
to BOLL expression during germ cell maturation in the human
fetal ovary does not occur in mice.
Discussion
In this report, we have examined the expression and
distribution of the RNA-binding proteins DAZL and BOLL during
human fetal oogenesis, revealing that whilst DAZL and BOLL
gene expression overlaps using gestational age as the
discriminator, DAZL and BOLL proteins are in fact expressed
PLOS ONE | www.plosone.org
8
September 2013 | Volume 8 | Issue 9 | e73996
DAZL and BOLL during Human and Mouse Oogenesis
Figure 6. Comparison of human and mouse DAZL/BOLL expression patterns during meiosis. A) DAZL and BOLL show
distinct spatio-temporal distributions which change during human fetal ovarian development. The timeline is depicted in stages of
meiosis in the human due to the wide range of germ cell developmental stages present at any specific week of gestation. DAZL is
expressed before and at meiosis initiation but down-regulated afterwards; BOLL is transiently expressed at later stages of meiosis
with minimal overlap with DAZL. DAZL is re-expressed in oocytes within primordial follicles. B) Expression of Dazl and Boll during
meiosis in the feto-neonatal mouse ovary. Dazl is expressed before and throughout meiosis, whereas Boll is detected only
transiently expressed around E15.5.
doi: 10.1371/journal.pone.0073996.g006
late meiotic prophase I. Finally, we sought to determine
whether the switch in expression from DAZL to BOLL that we
observe during human fetal oogenesis also occurs during germ
cell development in the fetal mouse ovary. We found that
although Boll expression in the fetal mouse ovary is also limited
to a narrow window during meiotic prophase I (as in humans),
Dazl is extensively co-expressed with Boll in the germ cells of
the fetal mouse ovary (Figure 6B). Thus in the mouse there is
continuous expression of Dazl throughout fetal germ cell
development with coincident brief expression of Boll, in
contrast to the human fetal ovary, wherein there is a transient
loss of DAZL expression at the time of BOLL expression, and
the co-expression of both proteins by the same germ cells is
rare.
Previous work from our laboratory reported developmental
changes in DAZL gene expression during human fetal
oogenesis, with transcript levels increasing substantially
between the first and second trimester [26]. Human ovarian
development during the second trimester encompasses two
PLOS ONE | www.plosone.org
key developmental stages; the formation of germ cell nests and
entry into and progression through meiotic prophase I
(approximately 12-16wga) and the breakdown of germ cell
nests and the assembly of individual oocytes into primordial
follicles (approximately 17wga onwards) [32]. The data
presented here refines that of Anderson et al. (2007), revealing
that the increase in DAZL expression occurs between 8-9 and
14-16 weeks gestation, co-incident with the onset of meiosis in
the human fetal ovary [33-35]. The expression of DAZL in
premeiotic and early meiotic germ cells supports a conserved
role for this protein in regulating the entry into meiosis in
humans and mice, where Dazl is required in fetal germ cells to
enable them to respond appropriately to the meiosis-inducing
signal retinoic acid [16]. The expression of BOLL overlaps with
that of DAZL, but the onset of expression is later, with BOLL
transcripts undetectable in the fetal ovary prior to the onset of
meiotic germ cell differentiation. The pattern of overlapping
DAZL and BOLL expression, with the onset of BOLL
expression occurring slightly later than that of DAZL, is very
9
September 2013 | Volume 8 | Issue 9 | e73996
DAZL and BOLL during Human and Mouse Oogenesis
similar to that reported previously in the fetal sheep ovary [24].
In that model, DAZL and BOLL transcripts are first detected at
38 days post coitum (dpc), but by 49dpc DAZL expression is
greater than that of BOLL, with both genes maximally
expressed at 56dpc, corresponding to the early stages of
meiotic prophase I. Interestingly, BOLL transcript levels, unlike
those of DAZL, appear to decline slightly between 14-16wga
and 17-20wga (although this does not reach statistical
significance). This likely reflects a progressive reduction of the
number of BOLL-positive germ cells in the fetal ovary at later
gestations, as oocytes downregulate BOLL and re-express
DAZL as they form primordial follicles, although this may result
from a dilution of BOLL mRNA due to the emergence of
additional BOLL-negative germ cells at this stage.
Although the patterns of DAZL and BOLL gene expression
overlap temporally, our data clearly demonstrate that the
spatial distribution of DAZL and BOLL proteins within the
human fetal ovary is quite distinct, with DAZL-expressing germ
cells towards the periphery, and BOLL-expressing germ cells
towards the centre of the ovary. This radial distribution of germ
cells reflects a gradient of differentiation, with smaller, less
mature germs in the peripheral cortex, and larger, more mature
germ less localised in the central medullary region [26,36,37].
Our finding that the nuclear diameter of BOLL-expressing germ
cells is greater than that of those expressing DAZL supports
this, and demonstrates that BOLL is expressed by germ cells at
later stages of development. This is consistent with previous
work from our laboratory, which showed that DAZL is
expressed in a stage specific fashion during human fetal
oogenesis, being downregulated as germ cells mature and
begin to express another germ cell-specific RNA binding
protein, VASA [26]. The transition from DAZL to VASA
expression was also found to occur in the germ cells of the
human fetal testis with increasing gestational ages [26],
revealing the existence of a germ cell maturation process
involving DAZL which is independent of meiosis. The relatively
sharp developmental switch from expression of DAZL to
expression of BOLL during human fetal oogenesis is distinct
from the overlapping patterns of expression of DAZL and
VASA, and coupled with the significant correlation of BOLL
expression with that of markers of meiosis, suggests the
existence of a parallel, meiosis-associated programme of germ
cell maturation involving DAZL and BOLL. This is supported by
the subsequent downregulation of BOLL, and reactivation of
DAZL expression that appears as germ cells form primordial
follicles, as follicle formation is dependent on oocytes reaching
the diplotene stage of meiotic prophase I and synaptonemal
complex disassembly [38]. The data presented here reveals a
complex relationship between DAZL, BOLL and meiosis during
human fetal ovarian development, and suggests that DAZL
may have meiosis-dependent and meiosis-independent roles
during human fetal germ cell development, the former being
restricted to the fetal ovary and the latter shared with the germ
cells of the human fetal testis.
An interesting observation of this study is the identification of
a subpopulation of germ cells that express SYCP3, but do not
express DAZL or BOLL. It is tempting to speculate that the
population of BOLL-negative, DAZL-negative, SYCP3-positive
PLOS ONE | www.plosone.org
germ cells identified here represents germ cells that are at a
stage of development immediately preceding follicle formation,
in which BOLL has been downregulated in anticipation of germ
cell nest disassembly and follicle formation, but which have not
yet proceeded far enough in the follicle assembly process to
have reactivated DAZL. However, these germ cells may
instead be destined for loss during the extensive waves of
apoptosis that occur prior to, and during, follicle formation
[32,39,40]. Determining the meiotic stage of the SYCP+-only
population of germ cells, coupled with assessment of coexpression of SYCP3 with proteins known to be expressed
around the time of follicle formation, may provide insight into
the developmental stage of these cells.
Zheng et al. (2011) recently reported the construction of a
mathematical model of the human fetal ovary, that integrates
microarray data on gene expression during human fetal ovarian
development with gene ontology and information on protein
function, and from this proposed that BOLL may function cooperatively with DAZL to prepare germ cells in the human fetal
ovary for entry into meiosis [41]. Whilst the microarray-derived
profiles of DAZL and BOLL gene expression in the human fetal
ovary used in their study display similarly overlapping
expression to our qPCR data, the immunohistochemical data
we present here disputes their model’s prediction, revealing
that BOLL is expressed in a different population of germ cells
to those expressing DAZL, and the onset of BOLL protein
expression occurs only well after meiosis has initiated,
precluding extensive physical interaction between the two
proteins. Importantly, this underlines the importance of relating
transcript data derived from whole organs comprised of
multiple different cell types and stages of development to the
spatial and temporal distributions of the transcripts and the
proteins they encode within the organ.
DAZL and BOLL are RNA-binding proteins, which act to
promote the translation of mRNA into protein by binding
specific motifs in the 3’ UTRs of target transcripts [5].
Numerous studies have identified putative mRNA targets of
DAZL [10,11,42-46], however only a limited number of these
have been validated in vivo [9,10]. In contrast, little is known
about the identity of the mRNA targets of BOLL [47], and
whether the motif to which it binds is similar to the GU-rich
sequences bound by DAZL remains unclear [48]. The
sequential expression of DAZL and BOLL during germ cell
maturation in the human fetal ovary, and the co-expression of
both proteins in the same germ cells in the fetal mouse ovary
around e15.5, raise interesting questions as to the identity of
the mRNA targets of these proteins, and to the degree of
overlap between them. Do DAZL and BOLL regulate at least a
number of the same transcripts, but at different developmental
stages in human fetal ovarian germ cell development? Or are
their targets distinct, as is might be expected if both proteins
are expressed in the same germ cells, as occurs during fetal
oogenesis in the mouse? The first possibility indicates that the
switch from DAZL to BOLL is largely functional compensation,
whereas the latter indicates that the two proteins may have
more distinct functions. The comparatively late spermiogenic
defect seen in Boll-deficient males [4,22] compared with the
earlier meiotic and pre-meiotic defects seen in Dazl-deficient
10
September 2013 | Volume 8 | Issue 9 | e73996
DAZL and BOLL during Human and Mouse Oogenesis
trimester fetal gonads was determined by PCR for the SRY
gene [51]. Extra-ovarian tissue was removed from dissected
ovaries, which were then either snap frozen on dry ice and
stored at -80°C (for subsequent RNA extraction), or fixed in
Bouins or 4% Neutral Buffered Formalin (NBF) for 2-3 hours for
processing into paraffin blocks for immunohistochemical
analysis.
mice on various genetic backgrounds [1,10,15-18,20,49],
supports the latter hypothesis. If this is true, then our data
suggest that DAZL and BOLL are required at distinct, relatively
non-overlapping developmental stages during human
oogenesis, with DAZL required to support the entry into, and
progression through the early stages of meiosis (and the
formation of primordial follicles), and BOLL required only during
established meiotic prophase I. This difference may be useful
in identifying the respective mRNA targets of DAZL and BOLL
during oogenesis, as comparison of the co-localisation of
DAZL, BOLL and the proteins encoded by their putative mRNA
targets during human fetal oogenesis will establish which
targets co-localise predominantly with one or the other. The
capacity of DAZ-family proteins to rescue deficiencies in these
genes across diverse species (exemplified by the partial rescue
of the spermatogenic defect in boule-deficient Drosophila by
human BOLL [50]) indicates that these proteins can recognise
and regulate the translation of the same mRNA targets, raising
the possibility that at least some mRNAs targets may be
regulated by both DAZL and BOLL at different stages of human
fetal oogenesis.
The data presented here raise important questions about the
validity of the Boll-knockout mouse as a mouse as a model for
understanding BOLL function during human female oogenesis.
It is tempting to speculate that the simultaneous co-expression
of Dazl and Boll in the germ cells of the fetal mouse ovary may
present a mechanism by which Dazl could compensate
functionally for the absence of Boll, permitting oogenesis to
proceed normally in mice. Indeed, such functional redundancy
has been proposed as one explanation for the relatively late
spermiogenic phenotype seen in Boll-/- male mice, which only
becomes manifest at a developmental stage after which Dazl is
no longer expressed [4]. The sequential expression of DAZL
and BOLL by distinct populations of human fetal germ cells at
different stages of development precludes the possibility of
functional compensation by DAZL in the absence of BOLL
during human fetal oogenesis, and raises the intriguing
possibility that BOLL mutations may contribute to fertility
defects in human females.
Animals
C57BL/6 mice were housed on a 12 hour light/dark cycle and
fed ad libitum according to UK Home Office and local
University of Edinburgh ethical standards. The day of vaginal
plug detection was designated as embryonic day (e) 0.5.
Gonads were isolated from fetuses at e13.5, e15.5 and e18.5,
and from neonatal mice on the day of birth (P0), and fixed in
4% NBF for 3 hours before processing into paraffin blocks.
RNA extraction, cDNA synthesis and qRT-PCR
Ethical approval for this study was obtained from Lothian
Research Ethics Committee (study code LREC 08/S1101/1).
All participants gave informed written consent in accordance
with national guidelines. Experiments involving mice were
approved by the University of Edinburgh Animal Research
Ethics Committee and performed according to the UK Animal
(Scientific Procedures) Act 1986.
RNA was extracted from 8-12wga human fetal ovaries using
the RNeasy Micro Kit (QIAGEN, Crawley, UK) and from
13-20wga ovaries using the RNeasy Mini Kit (QIAGEN)
according the manufacturer’s instructions. cDNA was
synthesized using the SuperScript VILO cDNA Synthesis Kit
(Life Technologies, Paisley, UK) following manufacturer’s
instructions. Identical reactions in which the Reverse
Transcriptase (RT) enzyme mix was replaced by nuclease-free
dH2O were prepared as negative controls.
For measurement of gene expression by qRT-PCR, standard
curves for DAZL, BOLL and RPL32 were generated by mixing
1µl serially diluted cDNAs (1/10, 1/25, 1/100, 1/250, 1/1000,
1/2500, 1/10000) with 0.2µl Forward/Reverse primers (25mM
stocks), 5µl SYBRGreen Master mix (Life Technologies) and
3.6µl nuclease-free dH2O, and run on ABI7900HT thermal
cycler (Life Technologies). Ct values were plotted against log
concentration, and the resulting slope for each was used to
calculate expression within each sample. To permit comparison
between individual samples, gene expression was calculated
relative to that of the housekeeping gene RPL32 (the
expression of which remains stable across the developmental
window examined). The PCR programme used was 50°C for 2
minutes (min), 95°C for 10min, then 40 cycles of 95°C for 15
seconds (sec), 60°C for 1min, and ended with a dissociation
stage (95°C for 15sec, 60°C for 15sec and 95°C for 15sec) to
check the specificity of PCR products. Quantitation of gene
expression in experimental samples was performed using the
same conditions as used for standard curves, but diluting the
cDNAs 1/10. Results were analysed using ABI SDS2.4
software, Microsoft Excel 2003 and GraphPad Prism 5
software. The sequences of the oligonucleotide primers used in
this study can be found in Table S1.
Collection of human fetal ovaries
Immunofluorescence
Human fetuses (8-20 weeks gestational age (wga)) were
obtained after elective termination of pregnancy. Terminations
were for social reasons, and all fetuses used in this study were
morphologically normal. Gestational age was determined by
ultrasound scan, and confirmed (for second trimester fetuses)
by direct measurement of foot length. The sex of the first
Human or mouse fetal ovaries were dissected, fixed in
Bouins or 4% NBF and processed into wax blocks by standard
methods [52]. 5µm sections were cut, mounted on glass slides
and dried overnight at 55°C. Slides were dewaxed in xylene
(2×5min) and rehydrated through graded alcohols (absolute
ethanol: 2×20sec; 90% ethanol: 20sec; 70% ethanol: 20sec).
Materials and Methods
Ethics statement
PLOS ONE | www.plosone.org
11
September 2013 | Volume 8 | Issue 9 | e73996
DAZL and BOLL during Human and Mouse Oogenesis
Supporting Information
Antigens were retrieved by pressure cooking in 250mL 0.05M
pH6 citrate buffer in a Decloaking Chamber (Biocare Medical,
CA, USA) set to programme: 125°C for 30sec, cool to 90°C,
10sec. A 30min incubation with 3% hydrogen peroxide diluted
in methanol or 10min incubation with Peroxidase Blocking
Reagent (DAKO, Glostrup, Denmark) was applied to block
endogenous peroxidase action. Tissues were blocked in
Phosphate Buffered Saline (PBS (Life Technologies))
containing 20% normal goat or rabbit serum (Diagnostics
Scotland, Carluke, UK), the species of serum used being the
same as that in which the secondary antibody was raised in)
and 5% Bovine Serum Albumin (BSA (Sigma Aldrich, Poole,
UK)) or Rodent Block (Mouse on Mouse Polymer IHC Kit
(Abcam, Cambridge, UK)) for 30min at room temperature, and
then incubated with 100µl primary antibody (see Table S2) per
slide in a humidified chamber overnight at 4°C. For negative
controls, serum block was applied in place of the primary
antibody. Next day, slides were incubated with 100µl
peroxidase-conjugated
secondary
antibody
at
room
temperature for 30min, and then with 50µl fluorescein Tyramide
Signal Amplification (TSA (PerkinElmer, Waltham, USA) for
10min. For single antigen detection, tissues were
counterstained using 4',6-diamidino-2-phenylindol (DAPI,
1/1000, diluted in PBS) for 10min, mounted and stored at 4°C.
For double-immunofluorescence, the primary antibody to the
first antigen was removed by microwaving the slides in 400ml
0.05M citrate buffer for 2.5min, cooled for 1 hour and then
blocked in serum block for 30min. The second primary antibody
incubation was performed exactly as for the first, as above. On
day three, tissues were stained with either peroxidaseconjugated secondary antibody and TSA (as on day 2), or with
a fluorophore-conjugated secondary antibody at room
temperature for 1h.
For triple-antigen immunofluorescence, the tissues were
microwaved and serum blocked again after detection of the
second primary antibody, and the third primary antibody
incubation performed as for the first and second ones. The
signal was detected as on day 3, and finally the tissues were
counterstained with DAPI. Images were captured using a 710
Confocal Microscope (Carl Zeiss, Oberkochen, Germany) and
Zen 2009 software.
Figure S1.
Validation of anti-DAZL and anti-BOLL
antibodies for immunofluorescence. HEK293 cells were
transfected with either pCMV6-DAZL or -BOLL vectors. AntiDAZL antibodies detected epitopes (green) in pCMV6-DAZL
transfected cells, but not in pCMV6-BOLL transfected or
untransfected cells (left and centre columns). Mouse anti-BOLL
antibody detected epitopes (green) in cells ectopicallyexpressing BOLL only (right column). Blue: DAPI. Scale bars:
20µm in all panels.
(TIF)
Figure S2. Specificity of DAZL and BOLL antibodies. Each
blot shows cell lysates of HEK293 cells either mock transfected
(transfection agent only) or transfected with vectors expressing
hDAZL (pCMV6-hDAZL) or hBOLL (pCMV6-hBOLL). α -tubulin
(detected as a band here of ~50kDa-55kDa; predicted
molecular weight: 50kDa) was used as loading control. A) and
B) Both anti-DAZL antibodies (mouse and rabbit as indicated)
detected a band at ~40-45kDa in only hDAZL-transfected cells
(consistent with the predicted molecular weight of DAZL plus
the Myc- and FLAG- tags of around 40kDa). C) anti-BOLL
antibody detected a band of ~39kDa only in hBOLL-transfected
cells. No comparable bands were found in mock transfected
cells.
(TIF)
Figure S3. Co-localisation of phospho-ATM with DAZL or
BOLL. A) immunofluorescent co-localisation of phospho-ATM
with DAZL or BOLL in the human fetal ovary (14 weeks
gestation). DAZL shows limited co-expression with phosphoATM, whereas almost all the phospho-ATM+ cells are also
BOLL+. Arrows indicate germ cells co-expressing of phosphoATM and DAZL or BOLL, and arrowheads indicate germ cells
expressing phospho-ATM only. Scale bars = 20µm. B)
Quantification of phospho-ATM co-localisation with DAZL and
BOLL. BOLL is expressed in ~80% of phospho-ATM+ cells,
which is significantly higher than the proportion of DAZL/
phospho-ATM double positive cells (37%; n=3, 14-16 week
human fetal ovaries, *p<0.05).
(TIF)
Germ cell diameter measurement and counting
Tiled images were taken for stained sections using 710
Confocal Microscope (Carl Zeiss) and Zen 2009 software, and
different areas include cortex and center of each image were
selected randomly for further analysis. For germ cell nuclear
diameter measurement, two perpendicular diameters of the
germ cell nucleus were measured using Image-Pro Plus
(Media Cybernetics, Silver Spring, USA), then averaged and
related back to a scale bar to convert pixels to μm. For cell
counting, the plugin cell counter of ImageJ (NIH, Maryland,
USA) was used and cell numbers were counted manually. The
data were analysed using Microsoft Excel 2003 and GraphPad
Prism 5 software. Data were analysed using t tests, or ANOVA
with Neumann-Keuls post hoc testing. P values less than 0.05
were considered statistically significant.
PLOS ONE | www.plosone.org
Methods S1. This file contains details of the methods
used to generate the results in Figures S1-S3, which are
not contained within the main body text of the article.
(DOCX)
Table S1. Oligonucleotide primers used for qRT-PCR.
(DOCX)
Table S2. Antibodies used for Immunofluorescence.
(DOCX)
Table S3. Antibodies used for Immunoblotting.
(DOCX)
12
September 2013 | Volume 8 | Issue 9 | e73996
DAZL and BOLL during Human and Mouse Oogenesis
Acknowledgements
Author Contributions
We are grateful to Anne Saunderson, Joan Creiger and the
staff of the Bruntsfield Suite of the Royal Infirmary of Edinburgh
for assistance with patient recruitment and specimen collection,
and to members of the Anderson and Childs laboratories for
helpful discussions throughout the project.
Conceived and designed the experiments: JH RAA AJC.
Performed the experiments: JH KS HK. Analyzed the data: JH
HK RAA AJC. Wrote the manuscript: JH RAA AJC.
References
1. Ruggiu M, Speed R, Taggart M, McKay SJ, Kilanowski F et al. (1997)
The mouse Dazla gene encodes a cytoplasmic protein essential for
gametogenesis. Nature 389: 73-77. doi:10.1038/37987. PubMed:
9288969.
2. Karashima T, Sugimoto A, Yamamoto M (2000) Caenorhabditis
elegans homologue of the human azoospermia factor DAZ is required
for oogenesis but not for spermatogenesis. Development 127:
1069-1079. PubMed: 10662646.
3. Eberhart CG, Maines JZ, Wasserman SA (1996) Meiotic cell cycle
requirement for a fly homologue of human Deleted in Azoospermia.
Nature 381: 783-785. doi:10.1038/381783a0. PubMed: 8657280.
4. VanGompel MJ, Xu EY (2010) A novel requirement in mammalian
spermatid differentiation for the DAZ-family protein Boule. Hum Mol
Genet 19: 2360-2369. doi:10.1093/hmg/ddq109. PubMed: 20335278.
5. Collier B, Gorgoni B, Loveridge C, Cooke HJ, Gray NK (2005) The
DAZL family proteins are PABP-binding proteins that regulate
translation in germ cells. EMBO J 24: 2656-2666. doi:10.1038/sj.emboj.
7600738. PubMed: 16001084.
6. Lee KH, Lee S, Kim B, Chang S, Kim SW et al. (2006) Dazl can bind to
dynein motor complex and may play a role in transport of specific
mRNAs. EMBO J 25: 4263-4270. doi:10.1038/sj.emboj.7601304.
PubMed: 16946704.
7. Kim B, Cooke HJ, Rhee K (2012) DAZL is essential for stress granule
formation implicated in germ cell survival upon heat stress.
Development 139: 568-578. doi:10.1242/dev.075846. PubMed:
22223682.
8. Maines JZ, Wasserman SA (1999) Post-transcriptional regulation of the
meiotic Cdc25 protein Twine by the Dazl orthologue Boule. Nat Cell
Biol 1: 171-174. doi:10.1038/11091. PubMed: 10559904.
9. Reynolds N, Collier B, Bingham V, Gray NK, Cooke HJ. (2007)
Translation of the synaptonemal complex component Sycp3 is
enhanced in vivo by the germ cell specific regulator Dazl. RNA 13:
974-981. doi:10.1261/rna.465507. PubMed: 17526644.
10. Reynolds N, Collier B, Maratou K, Bingham V, Speed RM et al. (2005)
Dazl binds in vivo to specific transcripts and can regulate the premeiotic translation of Mvh in germ cells. Hum Mol Genet 14:
3899-3909. doi:10.1093/hmg/ddi414. PubMed: 16278232.
11. Chen J, Melton C, Suh N, Oh JS, Horner K et al. (2011) Genome-wide
analysis of translation reveals a critical role for deleted in azoospermialike (Dazl) at the oocyte-to-zygote transition. Genes Dev 25: 755-766.
doi:10.1101/gad.2028911. PubMed: 21460039.
12. Yu YH, Lin YW, Yu JF, Schempp W, Yen PH (2008) Evolution of the
DAZ gene and the AZFc region on primate Y chromosomes. BMC Evol
Biol 8: 96. doi:10.1186/1471-2148-8-96. PubMed: 18366765.
13. Reijo R, Lee TY, Salo P, Alagappan R, Brown LG et al. (1995) Diverse
spermatogenic defects in humans caused by Y chromosome deletions
encompassing a novel RNA-binding protein gene. Nat Genet 10:
383-393. doi:10.1038/ng0895-383. PubMed: 7670487.
14. Saxena R, Brown LG, Hawkins T, Alagappan RK, Skaletsky H et al.
(1996) The DAZ gene cluster on the human Y chromosome arose from
an autosomal gene that was transposed, repeatedly amplified and
pruned. Nat Genet 14: 292-299. doi:10.1038/ng1196-292. PubMed:
8896558.
15. Lin Y, Page DC (2005) Dazl deficiency leads to embryonic arrest of
germ cell development in XY C57BL/6 mice. Dev Biol 288: 309-316.
doi:10.1016/j.ydbio.2005.06.032. PubMed: 16310179.
16. Lin Y, Gill ME, Koubova J, Page DC (2008) Germ cell-intrinsic and extrinsic factors govern meiotic initiation in mouse embryos. Science
322: 1685-1687. doi:10.1126/science.1166340. PubMed: 19074348.
17. Schrans-Stassen BH, Saunders PT, Cooke HJ, de Rooij DG (2001)
Nature of the spermatogenic arrest in Dazl -/- mice. Biol Reprod 65:
771-776. doi:10.1095/biolreprod65.3.771. PubMed: 11514340.
18. Saunders PT, Turner JM, Ruggiu M, Taggart M, Burgoyne PS et al.
(2003) Absence of mDazl produces a final block on germ cell
development at meiosis. Reproduction 126: 589-597. PubMed:
14611631.
PLOS ONE | www.plosone.org
19. Gill ME, Hu YC, Lin Y, Page DC (2011) Licensing of gametogenesis,
dependent on RNA binding protein DAZL, as a gateway to sexual
differentiation of fetal germ cells. Proc Natl Acad Sci U S A 108:
7443-7448. doi:10.1073/pnas.1104501108. PubMed: 21504946.
20. Haston KM, Tung JY, Reijo Pera RA (2009) Dazl functions in
maintenance of pluripotency and genetic and epigenetic programs of
differentiation in mouse primordial germ cells in vivo and in vitro. PLOS
ONE 4: e5654. doi:10.1371/journal.pone.0005654. PubMed: 19468308.
21. Xu EY, Moore FL, Pera RA (2001) A gene family required for human
germ cell development evolved from an ancient meiotic gene
conserved in metazoans. Proc Natl Acad Sci U S A 98: 7414-7419. doi:
10.1073/pnas.131090498. PubMed: 11390979.
22. Shah C, Vangompel MJ, Naeem V, Chen Y, Lee T et al. (2010)
Widespread presence of human BOULE homologs among animals and
conservation of their ancient reproductive function. PLOS Genet 6:
e1001022. PubMed: 20657660.
23. Kee K, Angeles VT, Flores M, Nguyen HN, Reijo Pera RA (2009)
Human DAZL, DAZ and BOULE genes modulate primordial germ-cell
and haploid gamete formation. Nature 462: 222-225. doi:10.1038/
nature08562. PubMed: 19865085.
24. Mandon-Pépin B, Oustry-Vaiman A, Vigier B, Piumi F, Cribiu E et al.
(2003) Expression profiles and chromosomal localization of genes
controlling meiosis and follicular development in the sheep ovary. Biol
Reprod 68: 985-995. PubMed: 12604652.
25. Vangompel MJ, Xu EY (2011) The roles of the DAZ family in
spermatogenesis: More than just translation? Spermatogenesis 1:
36-46. doi:10.4161/spmg.1.1.14659. PubMed: 22523742.
26. Anderson RA, Fulton N, Cowan G, Coutts S, Saunders PT (2007)
Conserved and divergent patterns of expression of DAZL, VASA and
OCT4 in the germ cells of the human fetal ovary and testis. BMC Dev
Biol 7: 136. doi:10.1186/1471-213X-7-136. PubMed: 18088417.
27. Reijo RA, Dorfman DM, Slee R, Renshaw AA, Loughlin KR et al. (2000)
DAZ family proteins exist throughout male germ cell development and
transit from nucleus to cytoplasm at meiosis in humans and mice. Biol
Reprod 63: 1490-1496. doi:10.1095/biolreprod63.5.1490. PubMed:
11058556.
28. Hartley PS, Bayne RA, Robinson LL, Fulton N, Anderson RA (2002)
Developmental changes in expression of myeloid cell leukemia-1 in
human germ cells during oogenesis and early folliculogenesis. J Clin
Endocrinol Metab 87: 3417-3427. doi:10.1210/jc.87.7.3417. PubMed:
12107261.
29. Roig I, Liebe B, Egozcue J, Cabero L, Garcia M et al. (2004) Femalespecific features of recombinational double-stranded DNA repair in
relation to synapsis and telomere dynamics in human oocytes.
Chromosoma 113: 22-33. PubMed: 15235794.
30. Hamer G, Kal HB, Westphal CH, Ashley T, de Rooij DG (2004) Ataxia
telangiectasia mutated expression and activation in the testis. Biol
Reprod 70: 1206-1212. PubMed: 14681204.
31. Pepling ME (2006) From primordial germ cell to primordial follicle:
mammalian female germ cell development. Genesis 44: 622-632. doi:
10.1002/dvg.20258. PubMed: 17146778.
32. Hartshorne GM, Lyrakou S, Hamoda H, Oloto E, Ghafari F (2009)
Oogenesis and cell death in human prenatal ovaries: what are the
criteria for oocyte selection? Mol Hum Reprod 15: 805-819. doi:
10.1093/molehr/gap055. PubMed: 19584195.
33. Childs AJ, Cowan G, Kinnell HL, Anderson RA, Saunders PT (2011)
Retinoic Acid signalling and the control of meiotic entry in the human
fetal gonad. PLOS ONE 6: e20249. doi:10.1371/journal.pone.0020249.
PubMed: 21674038.
34. Le Bouffant R, Guerquin MJ, Duquenne C, Frydman N, Coffigny H et
al. (2010) Meiosis initiation in the human ovary requires intrinsic retinoic
acid synthesis. Hum Reprod 25: 2579-2590. doi:10.1093/humrep/
deq195. PubMed: 20670969.
35. Bendsen E, Byskov AG, Andersen CY, Westergaard LG (2006)
Number of germ cells and somatic cells in human fetal ovaries during
13
September 2013 | Volume 8 | Issue 9 | e73996
DAZL and BOLL during Human and Mouse Oogenesis
36.
37.
38.
39.
40.
41.
42.
43.
the first weeks after sex differentiation. Hum Reprod 21: 30-35.
PubMed: 16155081.
Stoop H, Honecker F, Cools M, de Krijger R, Bokemeyer C et al. (2005)
Differentiation and development of human female germ cells during
prenatal gonadogenesis: an immunohistochemical study. Hum Reprod
20: 1466-1476. doi:10.1093/humrep/deh800. PubMed: 15734757.
Childs AJ, Kinnell HL, He J, Anderson RA (2012) LIN28 is selectively
expressed by primordial and pre-meiotic germ cells in the human fetal
ovary. Stem Cells Dev 21: 2343-2349. doi:10.1089/scd.2011.0730.
PubMed: 22296229.
Paredes A, Garcia-Rudaz C, Kerr B, Tapia V, Dissen GA et al. (2005)
Loss of synaptonemal complex protein-1, a synaptonemal complex
protein, contributes to the initiation of follicular assembly in the
developing rat ovary. Endocrinology 146: 5267-5277. doi:10.1210/en.
2005-0965. PubMed: 16150897.
Fulton N, Martins da Silva SJ, Bayne RA, Anderson RA (2005) Germ
cell proliferation and apoptosis in the developing human ovary. J Clin
Endocrinol Metab 90: 4664-4670. doi:10.1210/jc.2005-0219. PubMed:
15914527.
Tingen C, Kim A, Woodruff TK (2009) The primordial pool of follicles
and nest breakdown in mammalian ovaries. Mol Hum Reprod 15:
795-803. doi:10.1093/molehr/gap073. PubMed: 19710243.
Zheng P, Griswold MD, Hassold TJ, Hunt PA, Small CL et al. (2010)
Predicting meiotic pathways in human fetal oogenesis. Biol Reprod 82:
543-551. doi:10.1095/biolreprod.109.079590. PubMed: 19846598.
Fox M, Urano J, Reijo Pera RA (2005) Identification and
characterization of RNA sequences to which human PUMILIO-2
(PUM2) and deleted in Azoospermia-like (DAZL) bind. Genomics 85:
92-105. doi:10.1016/j.ygeno.2004.10.003. PubMed: 15607425.
Jiao X, Trifillis P, Kiledjian M (2002) Identification of target messenger
RNA substrates for the murine deleted in azoospermia-like RNAbinding
protein.
Biol
Reprod
66:
475-485.
doi:10.1095/
biolreprod66.2.475. PubMed: 11804965.
PLOS ONE | www.plosone.org
44. Maegawa S, Yamashita M, Yasuda K, Inoue K (2002) Zebrafish DAZlike protein controls translation via the sequence 'GUUC'. Genes Cells
7:
971-984.
doi:10.1046/j.1365-2443.2002.00576.x.
PubMed:
12296827.
45. Zeng M, Deng W, Wang X, Qiu W, Liu Y et al. (2008) DAZL binds to
the transcripts of several Tssk genes in germ cells. BMB Rep 41:
300-304. doi:10.5483/BMBRep.2008.41.4.300. PubMed: 18452650.
46. Zeng M, Lu Y, Liao X, Li D, Sun H et al. (2009) DAZL binds to 3'UTR of
Tex19.1 mRNAs and regulates Tex19.1 expression. Mol Biol Rep 36:
2399-2403. doi:10.1007/s11033-009-9470-1. PubMed: 19247806.
47. Urano J, Fox MS, Reijo Pera RA (2005) Interaction of the conserved
meiotic regulators, BOULE (BOL) and PUMILIO-2 (PUM2). Mol Reprod
Dev 71: 290-298. doi:10.1002/mrd.20270. PubMed: 15806553.
48. Jenkins HT, Malkova B, Edwards TA (2011) Kinked beta-strands
mediate high-affinity recognition of mRNA targets by the germ-cell
regulator DAZL. Proc Natl Acad Sci U S A 108: 18266-18271. doi:
10.1073/pnas.1105211108. PubMed: 22021443.
49. McNeilly JR, Saunders PT, Taggart M, Cranfield M, Cooke HJ et al.
(2000) Loss of oocytes in Dazl knockout mice results in maintained
ovarian steroidogenic function but altered gonadotropin secretion in
adult animals. Endocrinology 141: 4284-4294. doi:10.1210/en.
141.11.4284. PubMed: 11089564.
50. Xu EY, Lee DF, Klebes A, Turek PJ, Kornberg TB et al. (2003) Human
BOULE gene rescues meiotic defects in infertile flies. Hum Mol Genet
12: 169-175. doi:10.1093/hmg/ddg017. PubMed: 12499397.
51. Childs AJ, Kinnell HL, Collins CS, Hogg K, Bayne RA et al. (2010) BMP
signaling in the human fetal ovary is developmentally regulated and
promotes primordial germ cell apoptosis. Stem Cells 28: 1368-1378.
doi:10.1002/stem.440. PubMed: 20506112.
52. He J, Childs AJ, Zhou J, Anderson RA (2013) Immunohistochemical
approaches to the study of human fetal ovarian development. Methods
Mol Biol 957: 59-75. doi:10.1007/978-1-62703-191-2_4. PubMed:
23138944.
14
September 2013 | Volume 8 | Issue 9 | e73996